PsbD | D2 protein of PSII

KLH-comjugated synthetic peptide derived from the C-terminal of known PsbD sequences including Arabidopsis thaliana P56761, Hordeum vulgare P11849, Chlamydomonas reinhardtii P06007, Synechococcus sp. PCC 7002 P20898

Application example

2 µg of total protein from (1) Arabidopsis thaliana leaf extracted with Agrisera Protein Extraction Buffer, PEB (AS08 300), (2) Hordeum vulgare leaf extracted with PEB, (3) Chlamydomonas reinhardtii total cell extracted with PEB, (4) Synechococcus sp. 7942 total cell extracted with PEB, (5) Anabaena sp. total cell extracted with PEB were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 f

1 μg of chlorophyll from Pisum sativum (1), Zea mays, mesophyll (2) and bundle sheath (3), Echinochloa crus-galli, mesophyll (4) and bundle sheath (5), Arabidopsis thaliana (6) chloroplasts extracted with 0.4 M sorbitol, 50 mM Hepes NaOH, pH 7.8, 10 mM NaCl, 5 mM MgCl 2 and 2 mM EDTA were loaded to lanes. Samples were denatured with Laemmli buffer at 75°C for 5 min and were separated on 12% SDS-PAGE, and blotted 30 min to PVDF using wet transfer. Blot was blocked with 5% milk for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody Anti-PsbD at a dilution of 1: 5000 in 1% milk in TBS-T overnight at 4°C with agitation. The antibody solution was decanted and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG HRP conjugated, from Agrisera, AS09 602) diluted to 1:20 000 in 1 % milk in TBS-T for 1h at RT with agitation. The blot was washed 5 times for 5 min in TBS-T and 2 times for 5 min in TBS, and developed for 1 min with 1.25 mM luminol, 0.198 mM coumaric acid and 0.009% H 2 O 2 in 0.1 M Tris- HCl, pH 8.5. Exposure time in ChemiDoc System was 14 seconds.

Courtesy of Dr. Wioleta Wasilewska-Dębowska, University of Warsaw, Poland

Quantity control of chlorophyll amount loading onto gel. From 0,25 μg to 2,0 μg of chlorophyll from Pisum sativum chloroplasts were loaded to lanes. All steps of experiments were the same as described above.

Courtesy of Dr. Wioleta Wasilewska-Dębowska, University of Warsaw, Poland

There is a confirmed cross-reaction with TLA1 protein in Chlamydomonas reinhardtii.

For samples with a very low PSII content theremight be detection problems independent of the antibody. PSII proteins can vary in level depending upon liquid culture conditions. When the cells are in a stationary phase PSII content can drop to a very low level.

AS09 146S | PsbD | D2 protein of PSII protein standard for western blot quantitation and as a positive control
AS06 146PRE | PsbD | D2 protein of PSII, pre-immune serum, control for immunolocalization
AS05 084 | Anti-PsbA (D1) protein of PSII, C-terminal, rabbit antibodies
collection of antibodies to PSII proteins

D2 protein (PsbD) forms the reaction core of PSII (Photosystem II) as a heterodimer with the D1 protein (PsbA). PsbD is homologous to the D1 protein, with slightly higher molecular mass of about 39.5 kDa. Accumulation of D2 protein is an important step in the assemply of the PSII reaction centre complex.